U.S. patent application number 16/858810 was filed with the patent office on 2020-08-13 for drug composition for parenteral administration.
This patent application is currently assigned to THERABIOPHARMA INC.. The applicant listed for this patent is THERABIOPHARMA INC. KYOTO UNIVERSITY. Invention is credited to Tadashi Hashimoto, Atsushi Imaizumi, Hideaki KAKEYA, Masashi Kanai, Hitomi Ozawa, Nobuaki Takahashi.
Application Number | 20200254105 16/858810 |
Document ID | 20200254105 / US20200254105 |
Family ID | 1000004783051 |
Filed Date | 2020-08-13 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200254105 |
Kind Code |
A1 |
KAKEYA; Hideaki ; et
al. |
August 13, 2020 |
DRUG COMPOSITION FOR PARENTERAL ADMINISTRATION
Abstract
Provided is a curcumin pharmaceutical preparation that is highly
water soluble, can maintain the concentration of free curcumin in
the blood sufficiently high by being administered parenterally, can
effectively obtain a pharmacological action of curcumin, and is
highly safe. A pharmaceutical composition for parenteral
administration, including a water-soluble substance conjugate of
curcumin as an active component.
Inventors: |
KAKEYA; Hideaki; (Kyoto-shi,
JP) ; Kanai; Masashi; (Kyoto-shi, JP) ;
Takahashi; Nobuaki; (Kyoto-shi, JP) ; Hashimoto;
Tadashi; (Meguro-ku, JP) ; Imaizumi; Atsushi;
(Hino-shi, JP) ; Ozawa; Hitomi; (Yokohama-shi,
JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THERABIOPHARMA INC.
KYOTO UNIVERSITY |
Kawasaki-shi
Kyoto-shi |
|
JP
JP |
|
|
Assignee: |
THERABIOPHARMA INC.
Kawasaki-shi
JP
KYOTO UNIVERSITY
Kyoto-shi
JP
|
Family ID: |
1000004783051 |
Appl. No.: |
16/858810 |
Filed: |
April 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16313252 |
Dec 26, 2018 |
|
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|
PCT/JP2017/023753 |
Jun 28, 2017 |
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16858810 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 31/7028 20130101; A61K 47/549 20170801; A61P 37/08 20180101;
A61K 31/223 20130101; A61P 3/06 20180101; A61P 35/00 20180101; A61K
31/185 20130101; A61P 25/28 20180101; A61P 29/00 20180101; A61P
9/00 20180101; A61K 9/0019 20130101; A61K 31/12 20130101 |
International
Class: |
A61K 47/54 20060101
A61K047/54; A61K 31/12 20060101 A61K031/12; A61P 35/00 20060101
A61P035/00; A61P 29/00 20060101 A61P029/00; A61K 38/00 20060101
A61K038/00; A61K 31/223 20060101 A61K031/223; A61K 31/7028 20060101
A61K031/7028; A61P 37/08 20060101 A61P037/08; A61P 25/28 20060101
A61P025/28; A61P 9/00 20060101 A61P009/00; A61P 3/06 20060101
A61P003/06; A61K 31/185 20060101 A61K031/185; A61K 9/00 20060101
A61K009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2016 |
JP |
2016-128889 |
Claims
1-15. (canceled)
16. A method of administering curcumin to a subject, comprising
parenterally administering an aqueous solution comprising a
water-soluble curcumin conjugate.
17. The method of claim 16, wherein the water-soluble curcumin
conjugate is one or more conjugates with a compound selected from
the group consisting of glucuronic acid, sulfuric acid,
glutathione, and an amino acid.
18. The method of claim 16, wherein the water-soluble curcumin
conjugate is one or more conjugates with a compound selected from
the group consisting of glucuronic acid, sulfuric acid.
19. (canceled)
20. The method of claim 16, wherein the parenteral administration
is intravenous administration.
21. The method of claim 16, wherein the water-soluble curcumin
conjugate is curcumin monoglucuronide.
22. A method of maintaining a desired concentration of free
curcumin in the blood of a subject, comprising parenterally
administering a water-soluble curcumin conjugate.
23. The method of claim 22, wherein the water-soluble curcumin
conjugate is a conjugate with at least one member selected from the
group consisting of glucuronic acid, sulfuric acid, glutathione,
and an amino acid.
24. The method of claim 22, wherein the water-soluble curcumin
conjugate is a conjugate with at least one member selected from the
group consisting of glucuronic acid and sulfuric acid.
25. The method of claim 22, wherein the parenteral administration
is intravenous administration.
26. The method of claim 22, wherein the water-soluble curcumin
conjugate comprises curcumin monoglucuronide.
27. A method of treating a subject for at least one condition
selected from the group consisting of cancer, inflammation,
cholesterol, an allergy, a cognitive function, and heart disease
comprising parenterally administering an effective of amount of a
water-soluble curcumin conjugate.
28. The method of claim 27, wherein the parenteral administration
is intravenous administration.
29. The method of claim 27, wherein the water-soluble curcumin
conjugate comprises curcumin monoglucuronide.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a pharmaceutical
composition for parenteral administration represented by
intravenous administration.
BACKGROUND OF THE INVENTION
[0002] In recent years, it has been revealed that curcumin has a
pharmacological action such as a tumor formation inhibitory action,
an anti-oxidant action, an anti-inflammatory action, a
cholesterol-lowering action, an anti-allergic action, a brain
disease preventive action, and a heart disease preventive and
therapeutic action, and it has been studied to use the curcumin,
for example, for food (for example, functional food),
pharmaceuticals, or cosmetics.
[0003] However, it is known that curcumin is hardly soluble in
water, and therefore, is hardly absorbed in the body when orally
ingested as it is. In addition, it is indicated that most of the
curcumin that has been slightly absorbed by oral ingestion is
present in the blood as a curcumin conjugate conjugated with
glucuronic acid and/or sulfuric acid, and free form of curcumin is
present only slightly in the blood (Non Patent Literature 1).
[0004] In general, it is well known that in a case where foreign
substances are absorbed in the body, many of the foreign substances
are inactivated by in vivo metabolism such as conjugation. In order
to evaluate whether or not a curcumin conjugate has a
pharmacological action, A. Pal, et al. investigated the cell growth
inhibitory effect of a curcumin conjugate on KBM-5, Jurkat cells,
U266 and A549 cell lines in vitro, and found that free curcumin has
an effect of suppressing the growth of these cells (anti-cancer
effect), while indicating that there is no such an effect with the
curcumin conjugate (curcumin monoglucuronide and curcumin
diglucuronide) (Non Patent Literature 2). In addition, when
investigated the influence on the growth inhibitory effect of a
curcumin conjugate on human hepatocellular carcinoma cell lines
(HepG2 cells) and the gene expression, by oral administration,
Shoji, et al. found the cell growth inhibitory effect with free
curcumin, however, the effect was not found with the curcumin
conjugate (curcumin monoglucuronide), and indicated that as to also
the influence on the gene expression, the action was extremely low
with the curcumin conjugate (curcumin monoglucuronide) as compared
to that with free curcumin (Non Patent Literature 3). As described
above, it is considered that curcumin is also conjugated, as a
result of which a pharmacological action possessed by the curcumin
is inactivated.
[0005] For this reason, in order to sufficiently obtain the
expected pharmacological action of curcumin, it is required to
sufficiently increase the concentration of free curcumin in the
blood. However, due to the reasons described above, it is extremely
difficult to increase the concentration of free curcumin in the
blood even if curcumin is ingested orally.
[0006] Therefore, it is conceivable to administer curcumin
parenterally into the body, however, since curcumin is hardly
soluble in water, curcumin itself cannot be administered
parenterally. For this reason, for example, a method in which a
poorly water-soluble compound such as curcumin is subjected to a
treatment for increasing the solubility, and then the obtained
preparation is administered parenterally has been proposed (Patent
Literatures 1 and 2).
PRIOR ART
Patent Literature
[0007] Patent Literature 1: JP 5-178765 A [0008] Patent Literature
2: JP 2006-111534 A
Non Patent Literature
[0008] [0009] Non Patent Literature 1: Hui, et al., Clinical
Chemistry, 2009, 38: 59-68 [0010] Non Patent Literature 2: A. Pal,
et al., Bioorg Med Chem., 2014, 22(1), 435-439 [0011] Non Patent
Literature 3: M. Shoji, et al., Food Chemistry, 2014, 151,
126-132
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0012] However, in order to obtain a curcumin aqueous solution by
inclusion of curcumin with cyclodextrin, the amount of cyclodextrin
is required to be 100 to 1000 times the amount of the curcumin
(Patent Literature 2), and the curcumin aqueous solution was not
usable for parenteral administration.
[0013] Therefore, an object of the present invention is to provide
a curcumin preparation that is highly water soluble, can maintain
the concentration of free curcumin in the blood sufficiently high
by being administered parenterally, can effectively obtain a
pharmacological action of curcumin, and is highly safe.
Means for Solving the Problem
[0014] Therefore, the present inventors have conducted intensive
studies in order to solve the problem described above, it was found
that when a water-soluble substance conjugate of curcumin is
administered parenterally, surprisingly, the concentration of free
curcumin in the blood can be maintained at a high level, and as a
result, a pharmacological action possessed by curcumin can be
sufficiently obtained. In addition, the curcumin conjugate
contained in the pharmaceutical composition for parenteral
administration is an in vivo metabolite of curcumin, and therefore,
has extremely high safety.
[0015] That is, the present invention is to provide the following
[1] to [20].
[0016] [1] A pharmaceutical composition for parenteral
administration, including a water-soluble substance conjugate of
curcumin as an active component.
[0017] [2] The pharmaceutical composition for parenteral
administration according to [1], wherein the water-soluble
substance is one or more kinds selected from glucuronic acid,
sulfuric acid, glutathione, and amino acid.
[0018] [3] The pharmaceutical composition for parenteral
administration according to [1] or [2], wherein the water-soluble
substance is one or more kinds selected from glucuronic acid, and
sulfuric acid.
[0019] [4] The pharmaceutical composition for parenteral
administration according to anyone of [1] to [3], wherein the
pharmaceutical composition for parenteral administration is a
pharmaceutical composition selected from an anti-cancer agent, an
anti-inflammatory agent, a cholesterol-lowering agent, an
anti-allergic agent, a cognitive function improving agent, and a
heart disease preventive and therapeutic agent.
[0020] [5] The pharmaceutical composition for parenteral
administration according to any one of [1] to [4], wherein the
pharmaceutical composition for parenteral administration is a
pharmaceutical composition for intravenous administration.
[0021] [6] Use of a water-soluble substance conjugate of curcumin,
for producing a pharmaceutical composition for parenteral
administration.
[0022] [7] The use according to [6], wherein the water-soluble
substance is one or more kinds selected from glucuronic acid,
sulfuric acid, glutathione, and amino acid.
[0023] [8] The use according to [6] or [7], wherein the
water-soluble substance is one or more kinds selected from
glucuronic acid, and sulfuric acid.
[0024] [9] The use according to any one of [6] to [8], wherein the
pharmaceutical composition for parenteral administration is a
pharmaceutical composition for parenteral administration selected
from an anti-cancer agent, an anti-inflammatory agent, a
cholesterol-lowering agent, an anti-allergic agent, a cognitive
function improving agent, and a heart disease preventive and
therapeutic agent.
[0025] [10] The use according to any one of [6] to [9], wherein the
use is for producing a pharmaceutical composition for intravenous
administration.
[0026] [11] A water-soluble substance conjugate of curcumin, for
use as a medicine for parenteral administration.
[0027] [12] The water-soluble substance conjugate according to
[11], wherein the water-soluble substance is one or more kinds
selected from glucuronic acid, sulfuric acid, glutathione, and
amino acid.
[0028] [13] The water-soluble substance conjugate according to [11]
or [12], wherein the water-soluble substance is one or more kinds
selected from glucuronic acid, and sulfuric acid.
[0029] [14] The water-soluble substance conjugate according to any
one of [11] to [13], wherein the medicine for parenteral
administration is a medicine for parenteral administration selected
from an anti-cancer agent, an anti-inflammatory agent, a
cholesterol-lowering agent, an anti-allergic agent, a cognitive
function improving agent, and a heart disease preventive and
therapeutic agent.
[0030] [15] The water-soluble substance conjugate according to any
one of [11] to [14], wherein the medicine for parenteral
administration is a medicine for intravenous administration.
[0031] [16] A curcumin therapy, including parenterally
administering a water-soluble substance conjugate of curcumin.
[0032] [17] The curcumin therapy according to [16], wherein the
water-soluble substance is one or more kinds selected from
glucuronic acid, sulfuric acid, glutathione, and amino acid.
[0033] [18] The curcumin therapy according to [16] or [17], wherein
the water-soluble substance is one or more kinds selected from
glucuronic acid, and sulfuric acid.
[0034] [19] The curcumin therapy according to any one of [16] to
[18], wherein the curcumin therapy is a therapy selected from a
cancer therapy, an anti-inflammatory therapy, a
cholesterol-lowering therapy, an anti-allergic therapy, a cognitive
function improving therapy, and a heart disease preventive
therapy.
[0035] [20] The curcumin therapy according to any one of [16] to
[19], wherein the parenteral administration is intravenous
administration.
Effects of Invention
[0036] The pharmaceutical composition for parenteral administration
according to the present invention, in which a water-soluble
substance conjugate of curcumin is contained as an active
component, can maintain the concentration of free curcumin in the
blood at a high level for a long period of time by being
administered parenterally, and as a result, a pharmacological
action (for example, anti-tumor action) possessed by curcumin can
be effectively obtained. In addition, the pharmaceutical
composition for parenteral administration is a water-soluble
substance conjugate of curcumin, which contains an in vivo
metabolite of curcumin as an active component, and therefore, has
extremely high safety.
BRIEF DESCRIPTION OF DRAWINGS
[0037] FIG. 1 shows the changes in concentration in blood of each
of conjugate and free curcumin after intravenous administration of
curcumin conjugates.
[0038] FIG. 2 shows the anti-tumor effect of curcumin conjugates
(30 mg/kg) by intravenous administration.
[0039] FIG. 3 shows the anti-tumor effect of curcumin conjugates
(90 mg/kg) by intravenous administration.
[0040] FIG. 4 shows the influence of single dose of curcumin
conjugates on the body weight.
DESCRIPTION OF EMBODIMENTS
[0041] The pharmaceutical composition for parenteral administration
according to the present invention contains a water-soluble
substance conjugate of curcumin as an active component.
[0042] (A) Curcumin is a main component of the curcuminoid
contained in turmeric pigment, and is a compound represented by the
following structural formula (1).
##STR00001##
[0043] In the present invention, as the curcumin, chemically
synthesized curcumin may be used, or curcumin distributed as
turmeric pigment may be used. Examples of the turmeric pigment
include powdered turmeric obtained by pulverizing a dried rhizome
of Curcuma longa LINNE, crude curcumin or oleoresin (turmeric
oleoresin) obtained by extracting the powdered turmeric with an
appropriate solvent (for example, ethanol, fat and oil, propylene
glycol, hexane, or acetone), and purified curcumin.
[0044] In addition, curcumin includes both of keto and enol forms
of curcumin, which are tautomers.
[0045] As the water-soluble substance forming a conjugate with
curcumin, a water-soluble substance usually present in a living
body can be mentioned, and one or more kinds selected from
glucuronic acid, sulfuric acid, glutathione and amino acid are
preferred, and one or more kinds selected from glucuronic acid and
sulfuric acid are more preferred.
[0046] Herein, as the amino acid, an amino acid present in a living
body, for example, an essential amino acid can be mentioned.
[0047] The binding molar ratio of the curcumin in a curcumin
conjugate to the water-soluble substance is preferably
curcumin:water-soluble substance=1:1 to 1:3, more preferably
curcumin:water-soluble substance=1:1 to 1:2, and further more
preferably curcumin:water-soluble substance=1:1.
[0048] The conjugated form (binding form) of the curcumin and the
water-soluble substance is, for example, a form of the following
formula (2).
##STR00002##
[0049] (in the formula, at least one of R.sup.1 and R.sup.2 is a
residue of the water-soluble substance, and the remainder is a
hydrogen atom.)
[0050] In the formula (2), one or both of R.sup.1 and R.sup.2 are
preferably a glucuronic acid residue or a sulfuric acid residue,
and the remainder is preferably a hydrogen atom.
[0051] A water-soluble substance conjugate of curcumin can be
produced by the method described in Non Patent Literature 2 or
3.
[0052] The form of the pharmaceutical composition for parenteral
administration containing a water-soluble substance conjugate of
curcumin is not limited as long as it is administered parenterally,
and in particular, a composition for injection (injection) is
preferred. Examples of the composition for injection include a
composition for intravenous administration, and a composition for
subcutaneous administration, and a composition for intravenous
administration is more preferred.
[0053] The content of the water-soluble substance of curcumin in
the pharmaceutical composition for parenteral administration
according to the present invention is not particularly limited, and
is preferably 1 to 100% by mass, more preferably 5 to 100% by mass,
and furthermore preferably 10 to 100% by mass.
[0054] In the composition for parenteral administration according
to the present invention, in addition to the active components
described above, for example, water, a saline solution, a pH
adjusting agent, sugars, an acid, an alkali, a buffer agent, an
isotonizing agent, a stabilizer, an analgesic, and an antiseptic
agent can be mixed.
[0055] Herein, examples of the sugars include monosaccharides,
disaccharides, trisaccharides, polysaccharides, and a sugar
alcohol. Examples of the acid and the alkali include a
water-soluble inorganic acid, a water-soluble inorganic acid salt,
a water-soluble organic acid, a water-soluble organic acid salt, an
amino acid, and an amino acid salt.
[0056] In addition, the form of the composition for parenteral
administration according to the present invention may be a form of
a powder filler (crystal or freeze-dried product) to be dissolved
at the time of use, or may be a form of an aqueous solution.
[0057] As described in Examples later, the composition for
parenteral administration according to the present invention can
maintain the concentration in blood of free curcumin having a
pharmacological action at a high level over a long period of time,
for example, by being administered intravenously. In addition, the
composition for parenteral administration exhibits an excellent
pharmacological action (for example, anti-tumor effect) by being
administered parenterally, for example, being administered
intravenously, and therefore, is useful particularly as an
anti-cancer agent, an anti-inflammatory agent, a
cholesterol-lowering agent, an anti-allergic agent, a cognitive
function improving agent, or a heart disease preventive and
therapeutic agent, and in particular, is useful as an anti-cancer
agent.
EXAMPLES
[0058] Next, the present invention will be described in more detail
by way of Examples.
Production Example 1
(Preparation Method of Curcumin Conjugate (Curcumin
Monoglucuronide))
[0059] (1) 1.0 g (2.52 mmol) of acetobromo-.alpha.-D-glucuronic
acid methyl ester (compound a) and 326.0 mg (2.15 mmol) of vanillin
(compound b) were dissolved in 8.7 mL of quinoline, and into the
resultant mixture, 522.0 mg of silver oxide was added, then the
mixture was stirred for 30 minutes while cooling in ice, and then
the temperature of the mixture was raised to room temperature, and
the resultant mixture was stirred for 90 minutes. After 26.0 mL of
acetic acid was added into the resultant mixture, the mixture was
transferred to 260.0 mL of distilled water, and subjected to celite
filtration. The obtained water layer was subjected to extraction
twice with ethyl acetate, an organic layer was combined together,
and the resultant mixture was washed with brine and dried with
Na.sub.2SO.sub.4, and the solvent was distilled off under reduced
pressure. The obtained residue was purified with SiO.sub.2 flash
column chromatography (AcOEt/n-hexane=30:70, then 80:20), and 491.8
mg (49%) of a compound c was obtained.
##STR00003##
(2) 3.3 mL (32.07 mmol) of 2,4-pentanedione and 2.0 g (28.7 mmol)
of diboron trioxide were dissolved in 30.0 mL of ethyl acetate, and
the resultant mixture was stirred at 80.degree. C. for 30 minutes.
Into this resultant mixture, 2.2 g (14.4 mmol) of vanillin
(compound b) dissolved in 40.0 mL of ethyl acetate and 1.6 mL (5.96
mmol) of tributyl borate were added, and the mixture was stirred at
85.degree. C. for 30 minutes, and then into the resultant mixture,
0.5 mL (5.04 mmol) of n-butylamine was added dropwise, then the
temperature of the mixture was raised to 105.degree. C., and the
resultant mixture was stirred for 1.5 hours. Into the resultant
mixture, 10 mL of 1N hydrochloric acid was added, and the mixture
was stirred at 50.degree. C. for 1 hour, and then the temperature
of the mixture was returned to room temperature, the resultant
mixture was subjected to extraction twice with ethyl acetate, an
organic layer was combined together, and the resultant mixture was
washed with brine and dried with Na.sub.2SO.sub.4, and the solvent
was distilled off under reduced pressure. The obtained residue was
purified with NH--SiO.sub.2 (CHROMATOREX, manufactured by FUJI
SILYSIACHEMICAL LTD.) open column chromatography
(MeOH/CHCl.sub.3=1:30), and 1.31 g (39%) of a compound d was
obtained.
##STR00004##
(3) 483.0 mg (2.06 mmol) of the compound d and 214.9 mg (3.08 mmol)
of diboron trioxide were dissolved in 5.0 mL of ethyl acetate, and
the resultant mixture was stirred at 85.degree. C. for 30 minutes.
Into this resultant mixture, 805.0 mg (1.72 mmol) of the compound c
dissolved in 10.0 mL of ethyl acetate and 0.82 mL (3.05 mmol) of
tributyl borate were added, and the mixture was stirred at
85.degree. C. for 1 hour, and then into the resultant mixture, 71
.mu.L (0.72 mmol) of piperidine was added dropwise, and the mixture
was further stirred for 30 minutes. Into the resultant mixture, 7.0
mL of 0.5 N hydrochloric acid was added, and the mixture was
stirred at 50.degree. C. for 1 hour, and then the temperature of
the mixture was returned to room temperature, and the resultant
mixture was subjected to extraction twice with ethyl acetate, an
organic layer was combined together, and the resultant mixture was
washed with brine and dried with Na.sub.2SO.sub.4, and the solvent
was distilled off under reduced pressure. The obtained residue was
purified with SiO.sub.2 flash column chromatography
(AcOEt/n-hexane=60:40), and 917.7 mg (78%) of a compound e was
obtained.
##STR00005##
(4) In 24.5 mL of methanol, 875.1 mg (1.29 mmol) of the compound e
was dissolved, and into the resultant mixture, 24.5 mL of a 1 N
aqueous sodium hydroxide solution was added dropwise while cooling
in ice, and the mixture was stirred at 50.degree. C. for 1 hour.
The pH was adjusted to be 3 to 4 by using 50% formic acid, and the
precipitated solid was collected by filtration. The obtained solid
was purified with HPLC (PEGASIL ODS SP100, .PHI.20.times.250 mm,
eluent: CH.sub.3CN/H.sub.2O=45:55, 0.1% TFA), and 253.1 mg (36%) of
curcumin monoglucuronide (compound f) was obtained.
##STR00006##
Test Example 1
(Intravenous Administration Test)
(1) Test Animals
[0060] As the test animals, 9-week old SD rats (male, with a body
weight of 330 to 360 g, CHARLES RIVER LABORATORIES JAPAN, INC.)
were used.
(2) Intravenous Administration Method
[0061] The intravenous administration was performed as follows: a
predetermined amount of the curcumin monoglucuronide prepared in
Production Example 1 was dissolved into water for injection so as
to be 30 mg/kg; and the obtained preparation was administered
intravenously to each of the test animals (n=5) fasted for 12 hours
or more before the administration via an indwelling catheter in the
femoral artery of each of the test animals. In this regard, the
catheter was indwelled in the femoral artery under isoflurane
anesthesia on the day of administration and blood collection.
(3) Method for Collecting Blood and Plasma
[0062] Blood was collected via a catheter from the femoral artery
of each of test animals by using a heparin-treated syringe under
the restraint condition in a Ballman cage without anesthesia. In
this regard, around 0.6 mL of blood was collected after each lapse
of 1, 3, 5, 10, 15, 30, 60, 120, 240 and 480 minutes from the start
of intravenous administration, and the collected blood was
centrifuged (at 3,000 rpm, for 10 minutes, at 4.degree. C.) to
obtain the plasma.
(4) Fractional Determination of Concentration of Curcumin Conjugate
and Free Curcumin in Plasma
[0063] The concentration of each of the curcumin conjugate and free
curcumin in plasma was determined by the following methods.
a. Pretreatment
[0064] Into 20 .mu.L of the collected plasma, 100 .mu.L of a 0.1M
acetate buffer solution (pH 5.0) and 10 .mu.L of a
.beta.-glucuronidase solution (around 68,000 units/mL) or distilled
water were added, and the resultant mixture was kept at 37.degree.
C. for 1 hour, and then into the resultant mixture, 10 .mu.L of 50%
(v/v) methanol containing 20 ng/mL of mepronil, which was an
internal standard liquid, was added. Next, into the mixture, 0.5 mL
of chloroform was added, and the resultant mixture was stirred for
1 minute with a vortex mixer, then the mixture was treated for 15
minutes by using an ultrasonic generator, and the treated mixture
was fractionated into a chloroform layer and a water layer by
centrifugation (13,000.times.g, for 5 minutes, at room
temperature). Further, this fractionation process was repeated
twice. After that, the chloroform layer was collected, and dried
and solidified by distilling off the solvent with a vacuum
centrifugal concentrator, and to the dried and solidified matter,
100 .mu.L of 50% (v/v) methanol was added, and then the resultant
mixture was centrifuged (13,000.times.g, for 5 minutes, at room
temperature) to collect a supernatant liquid.
[0065] In this regard, in the following test, a supernatant liquid
prepared by using a .beta.-glucuronidase solution was used as an
enzyme-treated sample, and a supernatant liquid prepared by using
distilled water in place of the .beta.-glucuronidase solution was
used as a non-enzyme-treated sample.
b. Measurement Method
[0066] By analyzing 2 .mu.L of the enzyme-treated or
non-enzyme-treated sample prepared in the above-described "a.
Pretreatment" using LC-MS/MS (manufactured by Shimadzu
Corporation), the concentration of each of the curcumin conjugate
and free curcumin in plasma was determined.
[0067] That is, by measuring the concentration of the curcumin
contained in the enzyme-treated sample that had been obtained by
the treatment with .beta.-glucuronidase, the total concentration of
curcumin in plasma was measured.
[0068] On the other hand, by measuring the concentration of the
curcumin in the non-enzyme-treated sample that had not been treated
with .beta.-glucuronidase, the concentration of free curcumin in
plasma was measured.
[0069] Further, by subtracting the concentration of free curcumin
from the total concentration of curcumin, the concentration of the
curcumin conjugates contained in plasma was calculated.
[0070] In this regard, the LC-MS/MS analysis conditions were set to
be a LC column of Atlantis T3 (2.1.times.150 mm, 3 .mu.m,
manufactured by Waters), a column temperature of 40.degree. C., a
flow rate of 0.2 mL/min, and a mobile phase of A of a 0.1% formic
acid aqueous solution and B of 0.1% formic acid/acetonitrile, and
the gradient elution was performed under the following conditions
(Table 1). In addition, the MS analysis conditions were set to be
an ionization mode of Electron Spray thermo ionization (ESI),
Positive, a measurement mode of Multiple Reaction Monitoring (MRM),
curcumin of 369.1.fwdarw.177.2 (m/z), and mepronil of
270.fwdarw.119 (m/z), and the evaluation was performed.
[0071] Further, the calibration curve used for quantifying curcumin
was prepared with the measurement under the conditions similar to
those described above by using various kinds of standard solutions
(curcumin concentration of 0.9 to 225 ng/mL), which had been
prepared by adding 10 .mu.L of a 50% ethanol solution containing 20
ng/mL of mepronil into 90 .mu.L of a 50% (v/v) methanol solution
(curcumin standard solution) containing the curcumin in each amount
of 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 ng/mL.
TABLE-US-00001 TABLE 1 * Gradient elution conditions Time (min) 0
1.8 7 7.01 15 A (%) 40 5 5 40 40 B (%) 60 95 95 60 60
(5) Results
[0072] Concentration of each of the curcumin conjugates and free
curcumin in plasma in a case where the curcumin conjugates had been
administered intravenously was examined (FIG. 1).
[0073] The concentration of curcumin conjugates in plasma was
160492.+-.19156 ng/mL after the lapse of 1 minute from the
intravenous administration, 77803.+-.6442 ng/mL after the lapse of
3 minutes from the intravenous administration, 80489.+-.7161 ng/mL
after the lapse of 5 minutes from the intravenous administration,
46853.+-.10700 ng/mL after the lapse of 10 minutes from the
intravenous administration, 22918.+-.5271 ng/mL after the lapse of
15 minutes from the intravenous administration, 10191.+-.3502 ng/mL
after the lapse of 30 minutes from the intravenous administration,
4363.+-.1354 ng/mL after the lapse of 1 hour from the intravenous
administration, 1493.+-.371 ng/mL after the lapse of 2 hours from
the intravenous administration, 403.+-.120 ng/mL after the lapse of
4 hours from the intravenous administration, and 78.+-.39 ng/mL
after the lapse of 8 hours from the administration, and was reduced
with the passage of time. In addition, at this time, AUC 0.02-8 h
(ngh/mL) was 28387.+-.5093 ng/mL, and Cmax (ng/mL) was
160492.+-.19156 ng/mL.
[0074] On the other hand, it was found that the concentration of
free curcumin in plasma was not observed before the intravenous
administration of curcumin conjugates, however, was 10143.+-.3832
ng/mL after the lapse of 1 minute from the intravenous
administration of curcumin conjugates, and the free curcumin were
present at a high concentration in plasma. In addition, it was
found that the concentration of free curcumin in plasma was
7562.+-.2903 ng/mL after the lapse of 3 minutes from the
intravenous administration, 5707.+-.2539 ng/mL after the lapse of 5
minutes from the intravenous administration, 3596.+-.1233 ng/mL
after the lapse of 10 minutes from the intravenous administration,
2764.+-.796 ng/mL after the lapse of 15 minutes from the
intravenous administration, 1605.+-.592 ng/mL after the lapse of 30
minutes from the intravenous administration, 568.+-.197 ng/mL after
the lapse of 1 hour from the intravenous administration, 117.+-.39
ng/mL after the lapse of 2 hours from the intravenous
administration, 16.+-.5 ng/mL after the lapse of 4 hours from the
intravenous administration, and 6.+-.14 ng/mL after the lapse of 8
hours from the intravenous administration, and the free curcumin
was maintained at a high concentration in plasma for a long period
of time. In addition, at this time, AUC 0.02-8 h (ngh/mL) was
2778.+-.962 ng/mL, and Cmax (ng/mL) was 10143.+-.3832 ng/mL.
[0075] As described above, it was found that by administering
curcumin conjugates parenterally, the concentration of free
curcumin having a pharmacological action in blood can be maintained
at a high level over a long period of time.
Test Example 2
(Anti-Tumor Effect by Intravenous Administration of Curcumin
Conjugates in Various Doses)
(1) Test Animals
[0076] As the test animals, 5-week old BALB/cAnNcr j-nu/nu (homo)
mice (female, with a body weight of around 15 to 19 g, CHARLES
RIVER LABORATORIES JAPAN, INC.), which had been purchased and
acclimated to the environment for around 10 days, was used.
(2) Transplantation Method
[0077] 1.times.10.sup.7 or 4.times.10.sup.6 colorectal cancer cells
derived from adult human male colon HCT116 (ATCC No. CCL-247) were
transplanted subcutaneously into the flank of each of the test
animals by using a 27 G injection needle.
(3) Administration Method
[0078] Into each of the mice (n=5 or 8) inoculated with the cancer
cells (HCT116), after each lapse of 0, 2, 4, 6 and 8 days from the
start of inoculation or after each lapse of 0, 3, 5, 7, 10, 12, 14,
17, 19 and 21 days from the start of inoculation, 150 to 230 .mu.L
of the injection prepared by dissolving the curcumin
monoglucuronide that had been prepared in the above-described
Production Example 1 into water for injection (prepared so that
curcumin monoglucuronide was administered in an amount of 30 mg or
90 mg per kg body weight) was administered intravenously by using
an injection needle, and further the tumor size of each of the mice
was examined with the passage of days. In this regard, in the
control group, mice to which water for injection excluding curcumin
monoglucuronide had been administered intravenously were used.
(4) Evaluation of Tumor Size
[0079] For the tumor size, after each lapse of 0, 4, 7, 11 and 14
days from the start of administration or after each lapse of 0, 4,
7, 11, 14, 18 and 21 days from the start of administration, the
length, width and height of the tumor were measured by using a
vernier caliper, and the tumor size was calculated by the following
equation.
Tumor size (mm.sup.3)=length.times.width.times.height.times.0.5
(5) Concentration of Curcumin Conjugates and Free Curcumin in Tumor
Tissue
[0080] For the mice (n=8) inoculated with the 4.times.10.sup.6
cancer cells (HCT116), the tumor after the lapse of 2 hours from
the intravenous administration of curcumin monoglucuronide (90
mg/kg) after the lapse of 21 days from the start of inoculation was
removed from each of the mice, the removed tumor was rinsed with a
saline solution, and then immediately frozen in liquid nitrogen,
and the frozen tumor was stored in an ultra-low temperature tank at
-80.degree. C.
[0081] The frozen and stored tumor was thawed while cooling in ice,
an adequate amount of a saline solution was added to the thawed
tumor, and the thus obtained tumor was homogenized with a glass
homogenizer, the concentration of each of the curcumin conjugates
and free curcumin in the pulverized tumor obtained as described
above were measured to examine the concentration of each of the
curcumin conjugates and free curcumin contained in the tumor
tissue.
(6) Results
[0082] (Dose of 30 mg/kg--in Case of Mouse)
[0083] The anti-tumor effect was examined in a case where 30 mg of
curcumin conjugates (curcumin monoglucuronide) per kg body weight
of a mouse was administered parenterally (FIG. 2).
[0084] In a case where curcumin conjugates (curcumin
monoglucuronide) were administered parenterally (30 mg/kg--mouse),
the tumor size was 142.9.+-.45.5 mm.sup.3 after the lapse of 0 day
from the start of administration, 275.1.+-.88.1 mm.sup.3 after the
lapse of 4 days from the start of administration, 326.1.+-.110 0.7
mm.sup.3 after the lapse of 7 days from the start of
administration, 489.4.+-.147.8 mm.sup.3 after the lapse of 11 days
from the start of administration, and 581.2.+-.197.4 mm.sup.3 after
the lapse of 14 days from the start of administration.
[0085] On the other hand, in a case of the control group, the tumor
size was 142.5.+-.39.9 mm.sup.3 after the lapse of 0 day from the
start of administration, 297.0.+-.82.9 mm.sup.3 after the lapse of
4 days from the start of administration, 435.1.+-.138.6 mm.sup.3
after the lapse of 7 days from the start of administration,
653.3.+-.239.7 mm.sup.3 after the lapse of 11 days from the start
of administration, and 825.9.+-.271.3 mm.sup.3 after the lapse of
14 days from the start of administration, and showed larger values
at any time as compared to those in the case where the curcumin
conjugates (curcumin monoglucuronide) had been administered
parenterally.
[0086] In addition, when the biochemical test using the serum
obtained by the blood collection after the lapse of 14 days from
the start of administration was referred to Oriental Yeast Co.,
Ltd. (biochemical test set (liver and biliary diseases set), 21
items), no abnormality was found in the test values.
(Dose of 90 mg/kg--in Case of Mouse)
[0087] The anti-tumor effect was examined in a case where 90 mg of
curcumin conjugates (curcumin monoglucuronide) per kg body weight
of a mouse was administered parenterally (FIG. 3).
[0088] In a case where curcumin conjugates (curcumin
monoglucuronide) were administered parenterally (90 mg/kg--mouse),
the tumor size was 17.4.+-.3.2 mm.sup.3 after the lapse of 0 day
from the start of administration, 23.6.+-.8.4 mm.sup.3 after the
lapse of 4 days from the start of administration, 42.6.+-.16.6
mm.sup.3 after the lapse of 7 days from the start of
administration, 77.5.+-.28.9 mm.sup.3 after the lapse of 11 days
from the start of administration, 122.9.+-.42.6 mm.sup.3 after the
lapse of 14 days from the start of administration, 204.4.+-.76.2
mm.sup.3 after the lapse of 18 days from the start of
administration, and 190.2.+-.70.1 mm.sup.3 after the lapse of 21
days from the start of administration.
[0089] On the other hand, in a case of the control group, the tumor
size was 17.6.+-.2.9 mm.sup.3 after the lapse of 0 day from the
start of administration, 29.7.+-.5.9 mm.sup.3 after the lapse of 4
days from the start of administration, 61.8.+-.14.9 mm.sup.3 after
the lapse of 7 days from the start of administration, 106.3.+-.22.0
mm.sup.3 after the lapse of 11 days from the start of
administration, 179.8.+-.29.5 mm.sup.3 after the lapse of 14 days
from the start of administration, 376.4.+-.81.7=.sup.3 after the
lapse of 18 days from the start of administration, and
505.6.+-.100.4 mm.sup.3 after the lapse of 21 days from the start
of administration, and showed larger values at any time as compared
to those in the case where the curcumin conjugates (curcumin
monoglucuronide) had been administered parenterally.
[0090] In addition, when the concentration of each of the curcumin
conjugates and free curcumin contained in the tumor tissue was
examined, the concentration of the curcumin conjugates in the tumor
tissue was 605.4.+-.465.5 ng/g, and the concentration of the free
curcumin in the tumor tissue was 1464.5.+-.840.9 ng/g.
[0091] From these results, it was found that the curcumin
conjugates exerted the effect of significantly suppressing the
tumor growth by being administered parenterally. Further, it was
observed that free curcumin having a pharmacological action was
present in the tumor at a high concentration, and the tumor growth
inhibitory effect was presumed to be due to the free curcumin.
Test Example 3
(Single Dose Toxicity Test)
[0092] Intravenous administration of a single dose of curcumin
monoglucuronide was performed to each of the mice at each dose of
125, 250, 500 and 1000 mg/kg, and the toxicity thereof was
evaluated. In this regard, the number of mice in each group was set
to be n=5.
(1) Test Animals
[0093] As the test animals, 6-week old Slc: ICR mice (male, with a
body weight of 27 to 31 g, Japan SLC, Inc.), which had been
purchased and acclimated to the environment for around 6 days, was
used.
(2) Administration Method
[0094] 320 to 370 .mu.L of the solution prepared by dissolving the
curcumin monoglucuronide prepared in the above-described Production
Example 1 into a saline solution (in cases of 125, 250 and 500
mg/kg) or water for injection (in a case of 1000 mg/kg) (solution
being prepared so that curcumin monoglucuronide was administered in
an amount of 125 mg, 250 mg, 500 mg or 1000 mg per kg body weight)
was administered intravenously to each of the mice (n=5) by using
an injection needle.
(3) State Observation
[0095] Observation of the state of each of the mice was performed
by recording the type, extent, expression time, recovery time, and
death (death detection) time of all of the toxic signs observed
macroscopically.
(4) Observation Time
[0096] Observation was performed at a time immediately after
administration, at a time after each lapse of 30 minutes, 1, 2, 4
and 6 hours from the start of administration, and once a day from
the day following the date of administration (up to after the lapse
of 14 days from the start of administration). In this regard, in
the 1000-mg/kg administration group, acute symptoms were observed
immediately after administration, and therefore, observation was
frequently performed up to after the lapse of 1 hour from the start
of administration.
(5) Measurement of Body Weight
[0097] The body weight was measured by using an electronic balance
(UW4200S, Shimadzu Corporation) immediately before administration,
and after each lapse of 1, 3, 7, 10 and 14 days from the
administration.
(6) Blood Biochemical Test
[0098] Blood biochemical test was performed by the following
method. That is, mice were fasted for 4 hours or more after the end
of the mouse observation period (after the lapse of 14 days from
the start of administration), the fasted mice were subjected to
laparotomy under anesthesia by intraperitoneal administration of
pentobarbital sodium, and the blood was collected from the
abdominal aorta (the amount of the blood was 0.6 mL or more). Next,
the collected blood was anti-coagulated with heparin sodium, and
then the anti-coagulated blood was centrifuged (4.degree. C., 3000
rpm, for 10 minutes) to obtain the plasma, and transaminase (AST
and ALT) contained in the plasma was measured.
(7) Autopsy Method
[0099] After the end of the observation period (after the lapse of
14 days from the start of administration), autopsy was performed as
follows: blood was collected from each of the mice under anesthesia
with pentobarbital sodium; and then the mice were sacrificed by
exsanguination by cutting the abdominal aorta and the caudal vena;
each of the sacrificed mice was dissected according to a
pathological technique; and the cranium, the intrathoracic organs,
the intraperitoneal organs, and the tissues thereof were observed
macroscopically.
(8) Results
(State of Mouse by Macroscopy)
[0100] With regard to the state of a mouse in each administration
group, in the 125-mg/kg administration group, no abnormality was
found in the states of all of the mice (n=5) throughout the
observation period.
[0101] In the 250-mg/kg administration group, piloerection was
observed in 1 case (n=1) after the lapse of 1 hour from the start
of administration, however, the mouse in this case was recovered
later.
[0102] In the 500-mg/kg administration group, reduction of
locomotor activity was observed in all of the mice (n=5) from
immediately after administration to after the lapse of 4 hours from
the administration, loose stools were observed in all of the cases
(n=5) from after the lapse of 30 minutes to 1 hour from the start
of administration, and piloerection was observed in 3 cases (n=3)
from after the lapse of 30 minutes to 4 hours from the start of
administration, however, all of the symptoms were recovered
later.
[0103] In the 1000-mg/kg administration group, prone position and
reduction of locomotor activity were observed in all of the mice
(n=5) from immediately after administration to after the lapse of
30 minutes from the start of administration. In addition, bradypnea
was observed in 2 cases (n=2) immediately after administration and
in 1 case (n=1) after the lapse of 30 minutes from the start of
administration. Further, dyspnea was observed in 2 cases (n=2)
within 30 minutes from the start of administration. Furthermore,
paralytic gait was observed in 2 cases (n=2) within 30 minutes from
the start of administration and in 1 case (n=1) from after the
lapse of 30 minutes to 1 hour from the start of administration.
Moreover, death was observed in 4 cases (n=4) within 30 minutes
from the start of administration and in 1 case (n=1) from after the
lapse of 30 minutes to 1 hour from the start of administration.
[0104] From these results, it was found to be safe at a single dose
of 500 mg/kg or less, and found that 50% lethal dose (LD.sub.50)
was between 500 mg/kg and 1000 mg/kg.
(Changes in Body Weight)
[0105] Changes in the body weight of a mouse in each group are
shown in FIG. 4. In the 125-mg/kg administration group, all of the
animals showed a steady weight gain up to after the lapse of 14
days from the start of administration. Slight weight loss was
observed in 1 case (n=1) after the lapse of 1 day from the start of
administration in the 250-mg/kg administration group and in 3 cases
(n=3) after the lapse of 1 day from the start of administration in
the 500-mg/kg administration group, however, all of the animals
showed a steady weight gain up to after the lapse of 14 days from
the start of administration.
(Blood Biochemical Test)
[0106] For the blood biochemical test of a mouse in each group
(125-, 250- and 500-mg/kg administration groups), the AST levels in
the 125-, 250- and 500-mg/kg administration groups were 37.+-.4,
34.+-.3 and 37.+-.3 IU/L, respectively, and the ALT levels in the
125-, 250- and 500-mg/kg administration groups were 24.+-.6,
25.+-.4 and 34.+-.5 IU/L, respectively, and which showed normal
levels, respectively. In this regard, the test was not conducted in
the 1000-mg/kg administration group because all of the mice
died.
(Autopsy)
[0107] When autopsy was performed in each group (125-, 250- and
500-mg/kg administration groups), no abnormality was found in all
of the administration groups. In this regard, the autopsy was not
performed in the 1000-mg/kg administration group because all of the
mice died.
[0108] From these results, it was found to be safe at a single dose
of up to 500 mg/kg without finding any abnormalities.
Test Example 4
[0109] (Deconjugation of Curcumin Diglucuronide with
.beta.-Glucuronidase)
[0110] It was examined whether or not curcumin diglucuronide
(manufactured by SynInnova Laboratories Inc.) was deconjugated with
.beta.-glucuronidase. In this regard, as a control, curcumin
monoglucuronide was used for the examination.
(1) Test Sample
[0111] As the curcumin monoglucuronide, curcumin monoglucuronide
prepared in the above-described Production Example 1 was used. In
addition, as the curcumin diglucuronide, commercially available
curcumin diglucuronide (manufactured by SynInnova Laboratories
Inc.) was used.
(2) Pretreatment
[0112] Into 100 .mu.L of a 0.1 M acetate buffer solution (pH 5.0)
and 10 .mu.L of a .beta.-glucuronidase solution (around 68,000
units/mL), 10 .mu.L of curcumin monoglucuronide (2.81 .mu.g/mL,
n=3) or 10 .mu.L of curcumin diglucuronide (2.45 .mu.g/mL, n=3) was
added, and the resultant mixture was kept at 37.degree. C. for 1
hour, and into this mixture, 10 .mu.L of 50% (v/v) methanol
containing 20 ng/mL of mepronil, which was an internal standard
liquid, was added. Next, into the resultant mixture, 0.5 mL of
chloroform was added, and the mixture was stirred for 1 minute with
a vortex mixer, and then the stirred mixture was treated for 15
minutes by using an ultrasonic generator, and the treated mixture
was fractionated into a chloroform layer and a water layer by
centrifugation (10,000.times.g, for 5 minutes, at room
temperature). Further, this fractionation process was repeated
twice. After that, the chloroform layer was collected, and dried
and solidified by distilling off the solvent with a vacuum
centrifugal concentrator, and to the dried and solidified matter,
100 .mu.L of 50% (v/v) methanol was added, and then the resultant
mixture was centrifuged (10,000.times.g, for 5 minutes, at room
temperature) to collect a supernatant liquid, and the collected
supernatant liquid was taken as an enzyme-treated sample.
(3) Measurement Method
[0113] In 2 .mu.L of the enzyme-treated sample prepared in the
above (2), free curcumin was measured by the method described in
"b. Measurement method" in (4) in Test Example 1.
[0114] In this regard, the calibration curve used for quantifying
curcumin was prepared with the measurement under the conditions
similar to those described above by using a supernatant liquid
(curcumin concentration of 4.1 to 261.5 ng/mL), which had been
prepared as follows: into 100 .mu.L of a 0.1 M acetate buffer
solution (pH 5.0) and 10 .mu.L of a .beta.-glucuronidase solution
(around 68,000 units/mL), 100 .mu.L of a 50% (v/v) methanol
solution (curcumin standard solution) containing the curcumin in
each amount of 4.1, 16.3, 65.4 and 261.5 ng/mL and 10 .mu.L of a
50% ethanol solution containing 20 ng/mL of mepronil were added;
and the resultant mixture was subjected to the treatment by
chloroform and the solvent removal by distillation with a vacuum
centrifugal concentrator in a similar manner as in the above (2);
then into this mixture, 100 .mu.L of 50% (v/v) methanol was added;
and then the resultant mixture was centrifuged (10,000.times.g, for
5 minutes, at room temperature) to obtain the supernatant
liquid.
(4) Results
[0115] The concentration of the curcumin contained in the
enzyme-treated sample, which had been obtained by enzymatically
treating curcumin monoglucuronide with .beta.-glucuronidase, was
159.1.+-.6.1 ng/mL.
[0116] On the other hand, the concentration of the curcumin
contained in the enzyme-treated sample, which had been obtained by
treating curcumin diglucuronide with .beta.-glucuronidase, was
82.4.+-.0.8 ng/mL.
[0117] From the results described above, similar to the curcumin
monoglucuronide, free curcumin was also observed by treating the
curcumin diglucuronide with .beta.-glucuronidase, and therefore it
was found that the curcumin diglucuronide was deconjugated with
.beta.-glucuronidase. For this reason, it is considered that even
in a case where curcumin diglucuronide is administered
parenterally, the effect similar to that of curcumin
monoglucuronide can be obtained.
* * * * *